Nucleic acid anchoring system comprising covalent linkage of an oligonucleotide to a solid support

ABSTRACT

The anchoring system generally comprises a solid support and a chemical linking moiety useful for ether formation with another chemical moiety on a nucleic acid molecule. The present invention further contemplates methods for anchoring a nucleic acid molecule to a solid support via a covalent linkage. The anchoring system of the present invention is useful inter alia in construction of nucleic acid arrays, to purify nucleic acid molecules and to anchor nucleic acid molecules so that they can be used as templates for in vitro transcription and/or translation experiments and to participate in amplification reactions. The present invention is particularly adaptable for use with microspheres and the preparation of microsphere suspension arrays and optical fiber arrays. The anchoring system permits the generation of an anchored oligonucleotide for use as a universal nucleic acid conjugation substrate for any nucleic acid molecule or population of nucleic acid molecules. The present invention further provides a kit useful for anchoring nucleic acid molecules or comprising nucleic acid molecules already anchored to a solid support.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to an anchoring system fornucleic acid molecules. The anchoring system generally comprises a solidsupport and a chemical linking moiety useful for ether formation withanother chemical moiety on a nucleic acid molecule. The presentinvention further contemplates methods for anchoring a nucleic acidmolecule to a solid support via a covalent linkage. The anchoring systemof the present invention is useful inter alia in construction of nucleicacid arrays, to purify nucleic acid molecules and to anchor nucleic acidmolecules so that they can be used as templates for in vitotranscription and/or translation experiments and to participate inamplification reactions. The present invention is particularly adaptablefor use with microspheres and the preparation of microsphere suspensionarrays and optical fiber arrays. The anchoring system permits thegeneration of an anchored oligonucleotide for use as a universal nucleicacid conjugation substrate for any nucleic acid molecule or populationof nucleic acid molecules. The present invention further provides a kituseful for anchoring nucleic acid molecules or comprising nucleic acidmolecules already anchored to a solid support.

2. Description of the Prior Art

Reference to any prior art in this specification is not, and should notbe taken as, an aclcnowledgment or any form of suggestion that thisprior art forms part of the common general knowledge in any country.

The increasing sophistication of recombinant DNA technology is greatlyfacilitating research and development in a range ofbiotechnology-related industries.

Many manipulations involving nucleic acid molecules requireimmobilization strategies. One immobilization strategy involves the useof binding partners such as avidin and streptavidin. Whilst the lattersystem has been successfully employed in many nucleic acid anchoringsystems, it does have some limitations and does not enable the fullgamut of nucleic acid manipulations now available to be performed onsingle and mixtures of nucleic acid molecules. It is also subject tonon-specific binding thus limiting the accuracy of any immobilizationreactions.

In addition, there are difficulties in using linker systems likestreptavidin and avidin in automated and high throughput systems.

The immobilization procedure can be complex and involve the use ofexpensive reagents. There is a need, therefore, to develop a universalconjugation system for nucleic acid molecules.

In accordance with the present invention, a universal conjugation systemhas been developed for anchoring nucleic acid molecules to a solidsupport. The system of the present invention has a myriad of uses inmolecular biology including micro or macro nucleic acid arrays,capturing, purifying and/or sorting nucleic acid molecules, RNAproduction for RNAi and short, interfering RNA (si-RNA) applications andmicrosphere nucleic acid technology, especially for microengineeredstructures and nanoshells. The system may also be usefully employed inhigh throughput and/or automated systems. In particular, the presentinvention provides a re-usable anchoring system for nucleic acidmolecules.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO: 1), <400>2 (SEQ ID NO:2),etc. A summary of the sequence identifiers is provided in Table 1. Asequence listing is provided after the claims.

The present invention provides a conjugation system for target nucleicacid molecules. The conjugation system facilitates immobilization oranchoring of the target nucleic acid molecules to a solid phase. Thesolid phase may be any form of solid support including microspheres,microchips, beads, slides such as glass slides, microtiter wells anddipsticks amongst many others.

The solid support is generally selected on the basis of ease ofmanipulation, inexpensiveness, thermal stability and stability toaqueous and/or organic solvents.

Silica and methacrylate microspheres are particularly useful especiallyfor use in suspension arrays or optical fiber arrays.

The solid support is generally modified to include a chemical moietycapable of engaging in the formation of a covalent bond with anotherchemical moiety present on a nucleic acid molecule (the tagoligonucleotide). Any number of chemical moieties may be employed on thesolid support but in a preferred embodiment, the solid support comprisesa thiolated surface capable of engaging in covalent bond formation withan acryl group linked to the 5′ end of a tag oligonucleotide via aspacer between the 5′ base of the oligonucleotide and the active group.One preferred form of anchoring system is shown in FIG. 1.

The level of success in anchoring the tag oligonucleotide to the solidsupport is measured by annealing an oligonucleotide which iscomplementary to the tag oligonucleotide (referred to herein as the“α-tag”) optionally labeled with a reporter molecule such as but notlimited to 6-FAM. The annealing of the α-tag results, in a preferredembodiment, in a 3′ single-stranded overhang (or “sticky end”)comprising the tag oligonucleotide.

Any target nucleic acid molecule is then ligated to the tagoligonucleotide via a bridging oligonucleotide. The bridgingoligonucleotide comprises a sequence of nucleotides complementary to anucleotide sequence of the 3′ overhang portion of the tagoligonucleotide and a sequence of oligonucleotides complementary to a 5′end portion of a target nucleic acid molecule.

Accordingly, a target nucleic acid conjugating system is providedcomprising a solid support having a tag oligonucleotide covalently boundto the surface of the solid support, the tag oligonucleotide renderedpartially double-stranded by annealing an etag oligonucleotide to thetag oligonucleotide to provide a 3′ overhang single-stranded portion ofthe tag oligonucleotide to which is annealed a bridging oligonucleotidehaving a nucleotide sequence capable of hybridizing to the 5′ endportion of a target nucleic acid molecule. Conveniently, the bridgingoligo is removed from the support prior to becoming active.

In one embodiment, therefore, the present invention provides a universalnucleic acid anchoring system comprising the structure:—S(—T)_(p)wherein:

-   -   S is a solid support having a chemical moiety capable of        covalent bond formation with a second chemical moiety;    -   T is a tag oligonucleotide comprising single-stranded DNA having        said second chemical moiety linked via a spacer molecule to its        5′ end, said spacer comprising mc+n atoms, having from about 1        to about 100 atoms, where m is the number of repeats of a small        subunit, c is the number of atoms in each repeat, and n is the        number of atoms not in the repeat; said T further comprising a        bridging oligonucleotide having a nucleotide sequence        complementary to 3′ overhang nucleotides on the tag        oligonucleotide and a further nucleotide sequence complementary        to a nucleotide sequence on a 5′ end of a target nucleic acid        molecule;    -   wherein T may be represented p times on the solid support        wherein p is from about 1 to about 100,000.

In the above structure, the line “—” represents a covalent bond betweena solid support surface chemical moiety and the chemical moiety on thetag oligonucleotide.

The universal anchoring system of the present invention permits thegeneration of arrays of nucleic acid molecules. When the solid supportcomprises microspheres, the present invention permits the generation ofsuspension arrays. The anchored nucleic acid molecules may be subjectto, for example, mutation identification or other manipulations such asin vitro transcription and/or translation reactions.

The nucleic acid anchoring system, i.e. S(—T)_(p), may be re-used and,hence, only a single anchoring reaction need take place for virtuallyunlimited customizations via specific targets and bridges

The present invention further contemplates a method for anchoring atarget nucleic acid to a substrate, said substrate comprising:

-   -   (i) a solid support having a surface chemical moiety;    -   (ii) a tag oligonucleotide having a chemical moiety linked to        its 5′ end via a spacer comprising a molecule with mc+n atoms        wherein m is the number of repeats of a small subunit and c is        the number of atoms in each repeat and n is the number of atoms        not in the repeat wherein the latter chemical moiety is in        covalent bond formation with the chemical moiety on the surface        of the solid support;    -   (iii) a complementary (α) tag oligonucleotide sequence which has        hybridized to said tag oligonucleotide sequence such that there        is a single-stranded nucleotide sequence constituting a 3′        overhang of the tag oligonucleotide;    -   (iv) a bridging oligonucleotide having a complementary        nucleotide sequence to the nucleotide sequence of the 3′        overhang portion of the tag oligonucleotide and which bridging        oligonucleotide has hybridized to its complementary sequence on        the tag oligonucleotide leaving a single-stranded portion of the        bridging oligonucleotide which has a complementary nucleotide        sequence to the 5′terminal portion of said target nucleic acid        molecule;        wherein said method comprises contacting said target nucleic        acid molecule to said substrate for a time and under conditions        to permit hybridization of the 5′ portion of the nucleic acid        molecule to the single-stranded portion of the bridging        oligonucleotide and permitting ligase-mediated covalent bond        formation between said target nucleic acid molecule and the        substrate.

A spacer generally but not necessarily comprise carbon and oxygen basedmolecules or is a hydrocarbon molecule such as having from about 1 toabout 100 atoms, more preferably from about 18 to about 50 atoms andeven more preferably from about 24 to about 36 atoms is particularlyuseful.

The spacer molecule is conveniently an alkyl, alkenyl or an alkynylmolecule including a hydrocarbon molecule. Preferably, the spacer is alinear non-branched hydrocarbon although many other molecules may beemployed such as ethylene oxy (PEG) or one or more amino acids toseparate the oligonucleotide from the surface of the solid support aslong as they are inert in terms of the constructs intended application.A summary of sequence identifiers used throughout the subjectspecification is provided in Table 1.

TABLE 1 Summary of sequence identifiers SEQUENCE ID NO: DESCRIPTION 1nucleotide sequence of 5′-acrydite universal tag 2 nucleotide sequenceof PO4 complementary tag 3 nucleotide sequence of bridge oligonucleotide4 nucleotide sequence of PO4 target 5 nucleotide sequence of a targetsynthesized with 5′ PO4 6 nucleotide sequence of a terminal T3polymerase signal sequence 7 nucleotide sequence of target DNA sequence8 nucleotide sequence of 5′ overhang of common sequence of bridge 9nucleotide sequence of common sequence of bridge 10 nucleotide sequenceof a tag sequence (FIG. 3A) 11 nucleotide sequence of an α-tag sequence(FIG. 3B)

A list of terms used herein is provided in Table 2.

TABLE 2 Terms TERM DESCRPTION tag oligonucleotide or tag oligonucleotidemolecule anchored to a solid support face via a covalent bond between achemical moiety on the surface of the solid support and a chemicalmoiety conjugated to the oligonucleotide via a spacer molecule α-tagoligonucleotide molecule comprising a nucleotide sequence complementaryto the tag oligonucleotide sequence solid support form of solid phase;includes microspheres, microchips, beads and slides bridgingoligonucleotide oligonucleotide which bridges the tag oligonucleotideand the target nucleic acid molecule; the bridging oligonucleotide has anucleotide sequence complementary to a 3′ nucleotide sequence on tag andan end portion of the target nucleic acid molecule spacer a moleculecomprising a number of atoms and having the structure mc + n wherein mis the number of repeats, and c is the number of atoms in each repeatand n is the number of atoms not in the repeat target nucleic acid DNAor RNA target having a single-stranded molecule end portioncomplementary to part of the bridging oligonucleotide anchoring/anchoredjoining of two molecules via a covalent linkage chemical moiety achemical group capable of forming a covalent bond with another chemicalmoiety

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation of the three component linkerused to modify thiolated solid phase, especially silica microsphereactivated by silanization with 3-mercaptopropyl trimethoxysilane. Allcomponents are synthesized using standard phosphoramidite couplingchemistry. (A) Reactive group; (B) Spacer (18 atom spacer, with m=6; c=3and n=2 following designation of mc+n atoms in the spacer, (C) Tag DNAsequence. This component is variable and can be engineered for specificapplication; (D) Complete Tag Linker. This component serves the purposeof separating the DNA of interest (referred to as Target DNA in text)from the surface as well as provides a method for amplification ofTarget after molecular testing.

FIG. 2 is a representation of the process of two-step bead activationwith silane to produce a surface with a high density of exposed thiolgroups to create a tagged microsphere. Step (1): Raw silica beads arereacted with sulfur containing silane (3-Mercaptoprophyltrimethoxysilane (HS—CH₂—CH₂—CH₂—Si(Ome₃)). Step (2): Activated beadswith dense blanket of surface thiols are reacted with Tag linker (seeFIG. 1) to produce a bead with many thousands of covalently bounduni-directionally tethered DNA molecules.

FIG. 3 is a diagrammatic representation showing testing of conjugationefficiency. (A) Immobilized tag sequence; (B) α-tag: reverse complementto tag with 3′ FAM label; (C) approx. 10⁴ untreated silica microspheresprobed with 10 pMol α-tag; (D) approx. 10⁴ activated beads probed with10 pMol α-tag; (E) approx. 10⁴ beads with immobilized Tags probed with10 pMol α-tag. Fluorescence calculated on Becton-Dickinson FacsCalibur.

FIG. 4 is a diagrammatic representation showing ligase-mediatedcustomization Phosphorylated target DNA and bridge DNA is mixed withtagged microspheres, T4 DNA ligase, and ATP. After brief reaction atroom temperature, bridge and unincorporated targets are removed by heatand separation from the microspheres.

FIG. 5 is a graphical representation showing testing of ligationefficiency by the use of OLIGREEN (registered trademark). Customizedbeads (Tag+Target; M2 or left-most peak) and tagged beads (Tag only/notarget; M1 or right-most peak) were stained with a small amount ofOLIGREEN (registered trademark). Beads were run on Becton DickinsonFacsCalibur flow cytometer. Ligation efficiency is measured by testingthe ratio of M2 (tagged+target)/M1 (tag only). For this example, M2/M1is approximately 2.5, indicative of a successful ligation. M2/M1 valuesof <1.7 generally represent ligation efficiency of less than 80% oftarget DNA modified.

FIG. 6 is a graphical representation of optimal overhang length forligase-mediated conjugation to tagged microspheres. Bridgeoligonucleotides differing by only the number of 5′ bases in direct basepairing with target were tested by OLIGREEN (registered trademark)ligation assay (see FIG. 5). (A) 4 base pair overlap; (B) five base pairoverlap; (C) six base pair overlap. M1 or Marker 1 is the meanfluorescence of the tagged microsphere. M2 is the mean fluorescence ofthe tagged microsphere post ligation of target. The quantity M2/M1measures the relative gain in fluorescence and thus the amount of boundDNA. In this example, five and six base overlaps have greater ligationefficiency than four base overlap.

FIG. 7 is a graphical representation showing universal binding analogsfor general use bridges in ligation-mediated conjugation of taggedmicrospheres. All bridges had a common sequence of 5′-CXXXXXT [SEQ IDNO:8] CAT AGC TGT CCT-3′ [SEQ ID NO:9]. The 3′ italicized 12 bases werecommon to all bridges and hybridized to the 3′ 12 bases of theimmobilized tag. The underlined sequence CXXXXXT [SEQ ID NO:8}represents the variable 5′ overhang which hybridized to the targetsequence. The Xs represent nucleotide positions which were variable inthis test between the actual base and inosines, which are generalbinding base analogs. FIG. 7A represents the unsubstituted bridge. FIGS.7B-E represent increasing numbers of inosines in the bridges. A box inthe left side of each figure gives the tested sequence of the 5′overhang. In each experiment, 1×10⁵ beads with immobilized tags werereacted with 2.0 nMols of target oligonucleotide and appropriate bridgeas well as 20 units T4 DNA ligase, 1 mM ATP, 10 mM MgCl₂.

Reactions were carried out at room temperature for 15 minutes. UnligatedDNAs were removed by two 0.2 M NaOH washes. Beads with ligase mediatedconjugated products were assayed by OLIGREEN (registered trademark)binding assay using Becton Dickinson FacsCalibur. Ligation efficiency iscalculated as the sample fluorescence post binding (M2) divided by thecontrol mean fluorescence (M1). At least 200 events for each data pointwere collected.

FIG. 8 is a diagrammatic representation showing use of ligase-mediatedcustomized silica microspheres in solid phase PCR. This experiment isdivided into three sections. (A) description of the regions of the DNAto be amplified with labeled probes and their targets; (B) Controls toassess pre-PCR presence of immobilized sequences; (C) Post-PCR probes ofamplified sequences.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a nucleic acid anchoring system whichfacilitates ligase-mediated conjugation of a target nucleic acidmolecule to a solid support via a tag oligonucleotide which isconjugated to the solid support via a covalent bond between a chemicalmoiety resident on the solid support and another chemical moiety on thetag nucleic acid molecule.

A first aspect of the present invention, therefore, is a tagoligonucleotide anchored to a solid support.

Accordingly, one aspect of the present invention provides a solid phasecomprising a surface with a first chemical moiety capable ofparticipating in covalent bond formation with a second chemical moietyconjugated to a tag oligonucleotide wherein the tag oligonucleotide is asubstrate for ligase-mediated covalent bonding to a target nucleic acidmolecule.

In one embodiment, the chemical moiety on the surface of the solid phaseis capable of covalent bond formation with a tag-associated amine group,thiol group or acryl group.

Accordingly, another aspect of the present invention is directed to asolid phase comprising a surface with a first chemical moiety selectedfrom a carboxyl group, an amine group, and a thiol group, said firstchemical moiety capable of participating in covalent bond formation witha second chemical moiety selected from an amine group, a thiol group andan acryl group conjugated to an oligonucleotide with the proviso thatwhen the solid phase surface moiety is a carboxyl group then thecovalent bond forms with an amine group, when the surface moiety is athiol group the tag associated moiety is an acryl group or thiol group,or amine group linked via a heterobifunctional linker. The presentinvention extends, however, to chemical moieties capable of any form ofcovalent bond formation with any other chemical entity.

In one preferred embodiment, the chemical moiety on the surface of thesolid phase is a carboxyl group and such a group is capable of covalentbond formation with a number of chemical moieties but especially anamine group and when the solid phase chemical moiety is an amine groupor a thiol group several methods employing heterobifunctionalcrosslinkers allow covalent bond formation with an aminated or thiolatedtag oligonucleotide.

Accordingly, another aspect of the present invention is directed to asolid phase comprising either a surface carboxyl group capable ofparticipating in covalent bond formation with an amine group, or asurface encoded amine or thiol group conjugated to a tag oligonucleotidevia a crosslinker.

In a most preferred embodiment, the solid phase surface chemical moietyis a thiol group.

Most preferably, the chemical moiety conjugated to the tagoligonucleotide is an acryl group.

In this embodiment of the present invention, there is provided a solidphase comprising a surface thiol group capable of participating incovalent bond formation with an acryl group conjugated to a tagoligonucleotide.

The solid phase is preferably in the form of a solid support such as amicrosphere, bead, glass, ceramic or plastic slide, a dipstick or thewall of a vessel such as a microtiter well. The form of the solidsupport is not critical and may vary depending on the applicationintended. However, microspheres such as silica or methacrylatemicrospheres are particularly useful in the practice of the presentinvention, especially for use in suspension arrays or optical fiberarrays.

The selection of solid supports is conveniently based on ease ofmanipulation, level of expense, thermal stability and/or stability inaqueous and/or organic solvents.

In a particularly preferred embodiment, therefore, the present inventionis directed to microspheres having a thiolated surface capable ofparticipating in linker mediated or direct covalent bond formation witha chemical moiety selected from an amine group, a thiol group and anacryl group conjugated to a tag oligonucleotide.

Generally, any number of chemical moieties may be present or exposed onthe surface of the solid support and these may range from a few hundredto several thousand.

In a particularly preferred embodiment, there are from about 1 to about100,000 surface chemical moieties potentially involved in covalentbonding per solid support. This is particularly the case when the solidsupport is a microsphere. Conveniently, the microsphere comprises fromabout 500 to about 80000 or more conveniently from about 1000 to about80000 chemical moieties per bead. Examples include 1000, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000,15000, 16000, 17000, 18000, 19000, 20000, 21000, 22000, 23000, 24000,25000, 26000, 27000, 28000, 29000, 30000, 31000, 32000, 33000, 34000,35000, 36000, 37000, 38000, 39000, 40000, 41000, 42000, 43000, 44000,45000, 46000, 47000, 48000, 49000, 50000, 51000, 52000, 53000, 54000,55000, 56000, 57000, 58000, 59000, 60000, 61000, 62000, 63000, 64000,65000, 66000, 67000, 68000, 69000, 70000, 71000, 72000, 73000, 74000,75000, 76000, 77000, 78000, 79000 or 80000.

In relation to one preferred embodiment, therefore, the presentinvention provides microspheres each comprising from about 3000 to about80000 such as about 4000 to about 80000 or more particularly about 50000to about 80000 surface thiol groups per microsphere.

The tag oligonucleotide having the chemical moiety capable of covalentbond formation with the solid phase surface chemical moiety may compriseany nucleotide sequence although the nucleotide sequence would generallybe known. One particularly useful sequence is an RNA polymerase promoternucleotide sequence such as the T3 RNA polymerase promoter nucleotidesequence. The benefit of the latter in terms of lining DNA is theability to generate RNA transcripts. However, any oligonucleotide ofknown sequence may be employed. The term “oligonucleotide” is not to beviewed to any limiting extent and may comprise from about 10 base pairs(bp) to hundreds of bp.

It is convenient to ensure that after binding of the tag oligonucleotideto the solid phase that the tag oligonucleotide does not exhibitinterference with the solid support surface. Consequently, a spacermolecule is generally included between the chemical moiety and the 5′end of the tag oligonucleotide. A spacer generally but not necessarilycomprise carbon and oxygen based molecules or is a hydrocarbon moleculesuch as having from about 1 to about 100 atoms, more preferably fromabout 18 to about 50 atoms and even more preferably from about 24 toabout 36 atoms is particularly useful. Examples of the number of atomsin the spacer include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 10 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100.

The spacer may also be multiple repeats such as 2 x (18 atoms) spacersor 3 x (6 atoms) spacers. The length of the spacer is not critical formost applications as long as a crucial distance threshold between thebead surface and the active end of the DNA tag is maintained.

Consequently, another aspect of the present invention contemplates anisolated tag oligonucleotide comprising a chemical moiety capable ofcovalent bond formation with a chemical moiety on the surface of a solidphase, said first mentioned chemical moiety conjugated to said tagoligonucleotide via a spacer molecule having mc+n atoms wherein m is thenumber of repeats, c is the length of the repeat and n is the number ofatoms of the spacer molecule not contained in repeats.

Generally, m is from about 1 to about 12 such as 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11 or 12 and n is preferably 1 or from 0 to about 10 such as 0,1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Conveniently, mc+n is from about 1 to about 100 such as 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99 or 100. Advantageously, the atoms are carbon or oxygen atoms.

The spacer molecule is conveniently an alkyl, alkenyl or an alkynylmolecule including a hydrocarbon molecule. Preferably, the spacer is alinear non-branched hydrocarbon although many other molecules may beemployed such as ethylene oxy (PEG) or one or more amino acids toseparate the oligonucleotide from the surface of the solid support aslong as they are inert in terms of the constructs intended application.

The 5′ tag oligonucleotide chemical moiety is conveniently an aminegroup, a thiol group or an acryl group if the solid support surfacechemical moiety is a thiol group.

In a most preferred embodiment, the 5′ chemical moiety on the tagoligonucleotide is an acryl group.

In accordance with the above aspect of the present invention, the solidsupport is preferably a microsphere although any solid support may beemployed.

Accordingly, another aspect of the present invention provides a solidphase comprising a tag oligonucleotide anchored to the surface of saidsolid phase via a covalent bond between a chemical moiety on the surfaceof the solid phase and a chemical moiety conjugated to said tagoligonucleotide via a multi atom spacer having the structure mc+nwherein m is the number of repeats, c is the size of the repeat, and nis the number of atoms not included in the repeats.

As indicated above, mc+n is from about 1 to about 100.

As indicated above, the covalent bond is conveniently a thiol groupcovalently bonded to an acryl group or covalently bridged throughbifunctional linkers to tag encoded amines or thiols. Furthermore, thespacer molecule is preferably from about 1 to about 100 carbon atoms inlength.

Consequently, another aspect of the present invention comprises anarticle of manufacture having the structure:S—(mc+n)—[x₁x₂ . . . x_(p)]wherein:

-   -   S is a solid support;    -   m is the number of repeats;    -   c is a repeat of size c;    -   n is the number of atoms not included in repeats; ; and    -   [x₁x₂ . . . x_(p)] is a nucleotide sequence of nucleotides x₁x₂        . . . x_(p) wherein each of x₁x₂ . . . x_(p) may be the same or        different and the nucleotide length, p, is from 5 to about 200.

In the above formation, the schematic

represents a covalent bond such as, for example, an amide bond or athioether bond.

The oligonucleotide sequence, i.e. x₁x₂ . . . x_(p) is any knownsequence such as the T3 RNA polymerase promoter. The oligonucleotidesequence may also comprise an additional nucleotide sequence having, forexample, translation start signals, ribosome binding sites and aninitiating metiuonine (ATG) triplet.

It is particularly convenient to ensure or to measure successfulcovalent attachment of the tag oligonucleotide sequence to the solidphase. This can be accomplished by incorporating an internal fluorwithin the tag oligonucleotide sequence. This would give an immediateand simple test of amount of binding. However, this approach isoperationally limiting because in most instances, the internal fluorconfounds future applications by either interfering with data readout orby interfering by inhibiting the chemistry of the anchored system.

A preferred approach to measurement of amount of conjugated tagoligonucleotide is to prepare a labeled reverse complement to the tagoligonucleotide. Conveniently, the labeled oligonucleotide sequence iscomplementary to the 5′ end of the anchored tag oligonucleotidesequence. The label may be any suitable label such as 6-FAM. The 5′ endis generally phosphorylated.

Accordingly, another aspect of the present invention provides a solidphase comprising a tag oligonucleotide of known sequence anchoredthereto via a covalent linkage between a chemical moiety on the surfaceof the solid phase and a chemical moiety conjugated to the tagoligonucleotide via a molecule of mc+n atoms wherein m is the number ofrepeated atoms, c is the number of atoms in a repeat and n is the numberof atoms not in the repeat and wherein mc+n is from about 1 to about100, said solid phase further comprising a second oligonucleotidesequence annealed by base pairing to a complementary nucleotide sequenceon said first mentioned tag oligonucleotides resulting in an overhang atthe 3 end of either the tag oligonucleotide or its complementaryoligonucleotide.

Preferably, the second oligonucleotide sequence comprises a label and isused to measure the success or otherwise of the covalent anchoring ofthe first oligonucleotide sequence to the solid phase.

The preferred label is 6-FAM.

Preferably, the first oligonucleotide sequence overhangs at its 3′ endover the second oligonucleotide sequence.

As indicated above, the second oligonucleotide is labeled and, hence, itbecomes a convenient assay for the success or otherwise of covalentattachment of the first oligonucleotide to the solid phase. One skilledin the art will immediately recognize that there are many variations inorder to determine the extent of covalent linkage and that the presentinvention should not be only limited to one particular means.

The essence of this aspect of the invention is a solid phase having afirst tag oligonucleotide attached thereto via covalent linkage betweena first chemical moiety on the surface of the solid phase (e.g. acarboxyl group) and a second chemical moiety conjugated to the firstoligonucleotide via a spacer molecule of length mc+n atoms as definedabove and a second tag oligonucleotide, optionally labeled with areporter molecule capable of giving an identifiable signal, whichanneals to complementary nucleotide sequences on the firstoligonucleotide to provide, in a preferred embodiment, a 3′ overhang ofthe first tag oligonucleotide and wherein the 5′ end of the second tagoligonucleotide is phosphorylated.

The complementary oligonucleotide to the tag oligonucleotide is referredto herein as α-tag or the α-tag oligonucleotide.

The present invention provides, therefore, in one embodiment:

-   -   (i) a solid phase such as a microsphere, microchip or the sides        of a well in a microtiter plate; and    -   (ii) a tag oligonucleotide having a chemical moiety conjugated        to the oligonucleotide via a molecule of mc+n atoms as described        above; wherein the chemical moiety on the oligonucleotide is in        covalent bond formation with a chemical moiety on the surface of        the solid phase.

Again, as stated above, although a covalent linkage such as an amidebond or thioether bond is particularly useful in the practice of thepresent invention, it is but one of a whole myriad of covalent linkageswhich may be used in accordance with the present invention.

In general, the efficient production of a solid phase, especially on asurface with great stability, is difficult. In many systems, great careis required to ensure maximally efficient chemical reactions. Enzymaticmanipulations, on the other hand, are relatively easy and can beperformed in aqueous solutions, at moderate temperatures. The mainadvantage of the system described here is flexibility. Since thedifficult covalent linkage between tag and solid phase is only performedonce in a large stock, subsequent additions to the initial tag DNA isdone easily at any point in the future with virtually any desired targetDNA on whatever portion of the original stock required by a particularapplication.

The above solid support generally further comprises a secondoligonucleotide (α-tag) in complementary base pairing to the firstmentioned oligonucleotide (tag) such that there is optionally a label onthe 3′ end of the α-tag oligonucleotide and the 5′ end is phosphorylatedwherein the tag oligonucleotide overhangs the α-tag oligonucleotide atthe 3′ end of the tag oligonucleotide.

The next step is the generation of a bridge oligonucleotide whichenables anchoring of a target nucleic acid molecule to the tagoligonucleotide anchored to the solid phase.

The bridging oligonucleotide, in the case where the tag oligonucleotideoverhangs at its 3′end relative to the annealed a tag oligonucleotide,anneals in a direction where the bridge's 3″ end is reversecomplementary to the overhanging portion of the tag oligonucleotide Thebridge's 5′ end is thus a 5′ overhang of the tag: bridge double stranded(ds) DNA.

The 5′ end of the bridge is then reverse complementary to the 5′ endportion of a target nucleic acid molecule. Both the 5′ end of the targetnucleic acid molecule and the 5′ end of the labeled α-tagoligonucleotide (complementary to the anchored tag oligonucleotide) arephosphorylated. A ligase-mediated covalent attachment then formsanchoring the target nucleic acid molecule to the anchored tag via thebridging oligonucleotide.

Accordingly, in one embodiment, there is provided a substrate foranchoring a target nucleic acid molecule, said substrate comprising:

-   -   (i) a solid phase having a first chemical moiety on its surface;    -   (ii) a tag oligonucleotide comprising a second chemical moiety        in covalent bond formation with the first chemical moiety, said        second chemical moiety conjugated to the tag oligonucleotide via        a molecule of structure mc+n atoms as defined above;    -   (iii) an optionally labeled oligonucleotide reverse        complementary to the tag oligonucleotide; and    -   (iv) a bridging oligonucleotide having complementary based to        the 3′ overhang region of the tag oligonucleotide and        complementary bases to the 5′ end portion of the target nucleic        acid molecule wherein the target nucleic acid molecule is        anchored to the tag oligonucleotide via ligase-mediated        conjugation.

The bridging oligonucleotide may be part of the solid phase complexprior to anchoring of the target nucleic acid molecule or it may befirst added to and annealed to the target nucleic acid molecule prior toannealing to the tag oligonucleotide.

Yet in a further embodiment, the solid phase-tag oligonucleotidecomplex, the bridging oligonucleotide and the target nucleic acidmolecule are mixed together and subjected to ligation conditions.

The target nucleic acid molecule is specific for each conjugationexperiment. Generally, its initial 5-30 bases are complementary to thebases at the 5′ end of the bridging oligonucleotide. The 5′ end of thetarget nucleic acid molecule is generally phosphorylated. A minimum offive bases complementary between the target nucleic acid molecule andthe tag oligonucleotide is enough to enable ligation but generallyinsufficient to permit cross-hybridization, especially when multiplexinga large number of target molecules.

Yet another aspect of the present invention provides a universal nucleicacid anchoring system comprising the structure:—S(—T)_(p)wherein:

-   -   S is a solid support having a chemical moiety capable of        covalent bond formation with a second chemical moiety;    -   T is a partially double-stranded oligonucleotide comprising        single-stranded tag oligonucleotide having said second chemical        moiety linked via a spacer molecule to its 5′end, said spacer        comprising carbon atoms having the structure mc+n wherein m is        the number of repeats of length c, and n is the number of atoms        in the spacer molecule not included in the repeats and wherein        mc+n generally ranges from about 1 to about 100_(n); said tag        oligonucleotide further comprising a complementary        oligonucleotide (α-tag) annealed to the tag oligonucleotide to        provide a method of measurement of conjugation success a; said T        further comprising a bridging oligonucleotide having a        nucleotide sequence reverse complementary to the 3′ overhang        nucleotide sequence of the tag oligonucleotide and a further        nucleotide sequence complementary to a nucleotide sequence on        the 5′ end of a target nucleic acid molecule;    -   wherein T may be represented p times on the solid support        wherein p is from about 1 to about 100,000.

Still another aspect of the present invention contemplates a method forimmobilizing a target nucleic acid molecule to a partiallydouble-stranded tag oligonucleotide anchored to a solid support, saidmethod comprising ligating a phosphorylated 5′ end of the target nucleicacid molecule to a complementary single-stranded portion of the tagoligonucleotide under conditions to permit ligase-mediated covalent bondformation wherein said tag oligonucleotide is covalently anchored to thesolid support via covalent bond formation between a first chemicalmoiety on the surface of the solid support and a chemical moietyconjugated to the tag oligonucleotide via a molecule of structure mc+nas defined above and wherein the tag oligonucleotide is renderedpartially double-stranded by annealing a complementary oligonucleotideto the tag oligonucleotide leaving a single-stranded 3′ terminal portionof the tag oligonucleotide which is used to capture the target nucleicacid molecule via a bridging oligonucleotide.

The present invention further provides a kit useful in capturing and/oranchoring target nucleic acid molecules. The kit is conveniently inmulti-compartment form wherein a first compartment comprises a solidsupport such as microspheres or microchips having a surface chemicalmoiety. A second compartment comprises a tag oligonucleotide having achemical moiety capable of covalent bond formation with the surfacechemical moiety of the solid support and wherein the chemical moiety onthe tag is linked to the tag via a molecule of the mc+n structure asdefined above. A third compartment comprises a labeled complementary tagoligonucleotide and a fourth compartment comprises a bridgingoligonucleotide.

In an alternative, the kit may comprise a solid support having apartially double-stranded tag oligonucleotide anchored theretocomprising a single-stranded 3′ end portion. The kit may then have abridging oligonucleotide already attached to the single-stranded portionof the tag oligonucleotide or this may be maintained separately. Atarget nucleic acid molecule is then ligated to the tag oligonucleotidevia the bridge oligonucleotide.

The anchoring system of the present invention has many uses such as indeconvolution of complex mixtures of nucleic acid molecules, sorting ofnucleic acid molecules and for generation of microarrays, suspensionarrays and optical fiber arrays.

The system may also be adopted to facilitating in vitro transcriptionand/or translation and the transcription and/or translation productsassayed or used to screen for ligand or binding partners.

The anchoring system of the present invention may be fully or partiallyautomated and may be used for high throughput screening of targetnucleic acid molecules.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 Selection of Components of Anchoring Systems

1. Solid support

The physicochemical structure of the surface of the solid support is animportant consideration for the choice of chemical reactive moiety ofthe DNA to exploit for covalent attachment. The main attributes of thesurface are:

-   (a) ease of manipulation;-   (b) inexpensive;-   (c) stable in extremes of temperatures; and-   (d) stable in both aqueous and organic solvents.

Suitable surfaces include glass slides for solid microarrays and silicaand methacrylate microspheres for use in suspension arrays, opticalfiber arrays, or micromachined devices. The one favoured at the momentand representing the most common conjugation chemistry involves athiolated surface is exemplified below.

2. A Universal Tag for Initial Modification of the Surface

In the present system, a reactive end (amine, thiol or acryl group) isused at the 5′ end of the DNA oligonucleotide. In the example givenhere, the 5′ reactive group is an acryl, followed by two —(OCH₂CH₂)₆spacers. These additions are made at point of synthesis.

To this common 5′ end architecture is added a 20 base linker designed onthe T7, T3, or SP6 RNA polymerase promoters along with an additional 18bases comprising transcription and translation start signals.

The universal tag comprises the structure:

[SEQ ID NO:1] 5′-Acrydite-C18-C18-TAATACGACTCACTATAGGGCGA3. A Labeled α-tag

To assay the successful covalent attachment of the tag to the surface, alabeled reverse complementary 16-mer built to bind to the first 16 basesof the tag is used. The 3′ end is fluoresceinated with 6-FAM and the 5′end is phosphorylated.

The sequence of the complementary tag is as follows:-

5′PO4-ATAGTGAGTCGTATTA-FAM [SEQ ID NO:2]4. A Bridge Oligo

This bridge is built to be complementary to the last six bases of thetag as well as the first five bases of the target. It is kept small foreasy removal from reactions, but long enough to be easily scored byelectrophoresis. The bridge needs no 5′ modifications.

Its structure is:-

5′-TCCCGCTCCTAGA [SEQ ID NO:3]5. Phosphorylated Target

This DNA is made to be specific for each experiment. It has its initialfive bases reverse complementary to the five 5′ bases of the bridge. The5′ end of the target is phosphorylated. The five bases of the targetwhich hybridise to the 5′ end of the bridge are sufficient to enableligation, but not sufficient enough to significantly add tocross-hybridization. In the present system test, the 3′ end of thetarget contained the reverse complement of the SP6 RNA polymerasepromoter allowing for either translation or, in concert with T7promoter, PCR amplication.

An example of a target is as follows:

[SEQ ID NO:4] 5′PO4-GGATCTGACACGGACTGATGAATTCC-α-sp6-3′

EXAMPLE 2 System Set-Up

1. Tag is Conjugated to Surface

The execution of this step depends on the chemistry and surface used.The assay for measurement of amount of covalent binding is performed bybinding α-tag to the solid surface. Amount of fluorescence at 521 nm ismeasured after excitation by a high-energy light source. The argon ionlaser of the ABI 377, ABI 3700 or BD FacsCalibur may conveniently beused to measure this quantity.

2. Target is Ligated to Tag by Bridging Ligation

The bridge and target are added in equimolar amounts to the tag-modifiedsurface with T4 DNA ligase. Successful ligation of target to tag ismeasured indirectly by measuring the ligation of α-tag to bridgeelectrophoretically (a 27-mer vs an 11-mer and a 16-mer) or by measuringthe binding of OLIGREEN, a single stranded fluorescent binding dye fromMolecular Probes. By measuring the amount of binding to the surfacebefore and after ligation, it is easy to quantify the amount of ssDNAgained by the ligation-anchoring step.

EXAMPLE 3 Universal Primed Target Production

The primary aim of this Example is to introduce a high efficiency, lowcost, easily used microsphere based system for capturing nucleic acidmolecules. The present system is useful for specific testing of reagentswhich can be used in conjunction with a flow cytometer or other beadbased instrument.

The system may also be used for generation of capture reagents forcombinatorial screening as well as a system for solid phase PCR and/orsingle-stranded extensions.

The three component linker used to modify a thiolated solid phase isshown in FIG. 1.

A Universal Forward Oligo (UF) is then generated and in one examplecomprises the SP6 RNA polymerase promoter with a 5′ acrydite, a 30-atomspacer, followed by the sequence. This is conjugated to form a bead: UFcomplex (FIG. 2).

The efficiency of conjugation is measured by measuring the binding ofα-UFO which is a phosphorylated, internally labeled complement to thefirst 13 bases of the UFO.

The resulting bead has a configuration shown in FIG. 3.

Successfully conjugated bead preps are made in bulk, 5×10¹⁰ beads(usually 5×10⁶ beads/ul, so 5×10⁹ beads/ml=about 10 ml of bead stock).

Specific targets are produced by a two step ligation protocol in which aUniversal Bridging Oligo (UBO) is first bound to the 5′ end of eachtarget and then the resulting “sticky-ended” target is ligated to thebead: UFO:α-UFO defined as above.

The UBO has the following characteristics. The first six bases 5′ willbe complementary to the last six bases of the UFO and the final fivebases will be random. The resulting complex is shown in FIG. 4.

Thus, a small (e.g. 1024) library is created. The key to this systemworking is the randomness of this library as well as the workable size.The size of this variable domain is kept at five to be both manageableas well as easily removed by gel filtration at the end of the firsthybridization step.

The target DNA is synthesized with a 5′ phosphate, a number of basesspecific to the experiment, and a terminal 17 bases complementary to theT3 RNA polymerase promoter. As an example, a target of sequenceGCAACCATTATC [SEQ ID NO:5] is synthesized with a 5′ PO₄ and a terminalT3 polymerase signal sequence of TCCCTTTAGTGAGGGTT [SEQ D NO:6] for thefollowing final construct:

To assess the efficiency of the ligation, the relative amounts of bound13-mer and 24-mer would be ascertained by quantitative capillaryelectrophoresis on an ABI 3700 analyzed with Genescan software.Populations of particles from successful ligations would be sorted byflow cytometry.

EXAMPLE 4 Ligase-Mediated Customization

Phosphorylated target DNA and bridge DNA is mixed with taggedmicrospheres, T4 DNA ligase, and ATP. After brief reaction at roomtemperature, bridge and unincorporated targets are removed by heat andseparation from the microspheres. A diagram of ligase-mediatedcustomization is shown in FIG. 4.

EXAMPLE 5 Ligation-Mediated Customization Efficiency

Customized beads (tag+target; M1 or left-most peak of FIG. 5) and taggedbeads (Tag only/no target; M2 or right-most peak) were stained with asmall amount of OLIGREEN (registered trademark). Beads were run onBecton Dickinson FacsCalibur flow cytometer. Ligation efficiency ismeasured by testing the ratio of M2 (tagged+target)/M1 (tag only). Forthis Example, M2/M1 is approximately 2.5, indicative of a successfulligation. M2/M1 values of <1.7 generally represent ligation efficiencyof less than 80% of target DNA modified. The results are shown in FIG.5.

EXAMPLE 6 Test of Optimal Overhang Length for Ligase-MediatedConjugation to Tagged Microspheres

Bridge oligonucleotides differing by only the number of 5′ bases indirect base pairing with target were tested by OLIGREEN (registeredtrademark) ligation assay (see FIG. 6). In this Figure, M1 or Marker 1is the mean fluorescence of the tagged microsphere. M2 is the meanfluorescence of the tagged microsphere post-ligation of target. Thequantity M2/M1 measures the relative gain in fluorescence and thus theamount of bound DNA. In this Example, five and six base overlaps havegreater ligation efficiency than four base overlap. The results areshown in FIG. 6.

EXAMPLE 7 Universal Binding Analogs for General use Bridges inLigation-Mediated Conjugation of Tagged Microspheres

All bridges had a common sequence of 5′-CXXXXXT [SEQ ID NO:8] CAT AGCTGT CCT-3′ [SEQ ID NO:9]. The 3′ italicized 12 bases were common to allbridges and hybridized to the 3′ 12 bases of the immobilized tag. Theunderlined sequence CXXXXXT [SEQ ID NO:8} represents the variable 5′overhang which hybridized to the target sequence. The Xs representnucleotide positions which were variable in this test between the actualbase and inosines, which are general binding base analogs. FIG. 7Arepresents the unsubstituted bridge. FIGS. 7B-E represent increasingnumbers of inosines in the bridges. A box in the left side of eachfigure gives the tested sequence of the 5′ overhang. In each experiment,1×10⁵ beads with immobilized tags were reacted with 2.0 nmols of targetoligonucleotide and appropriate bridge as well as 20 units T4 DNAligase, 1 mM ATP, 10 MM MgCl₂. Reactions were carried out at roomtemperature for 15 minutes. Unligated DNAs were removed by two 0.2 MNaOH washes. Beads with ligase mediated conjugated products were assayedby OLIGREEN (registered trademark) binding assay using Becton DickinsonFacsCalibur. Ligation efficiency is calculated as the samplefluorescence post binding (M2) divided by the control mean fluorescence(M1). At least 200 events for each data point were collected. Theresults are shown in FIG. 7.

EXAMPLE 8 Use of Ligase-Mediated Customized Silica Microspheres in SolidPhase PCR

This Experiment is divided into three sections. (A) A 187 bp DNAfragment generated by PCR with the following landmarks. (B) A taggedmicrosphere is customized with the phosphorylated forward primer. TheFAM labeled α-tag probe as well as the Cy5 complement to the target areused to assess the efficiency of the conjugation. Background levels ofbinding are determined for all labeled probes. (C) PCR is performedusing immobilized forward primer. Success is determined by strippingnon-covalently bound strand and reprobing with either the originalreverse primer or the internally labeled complement. The results areshown in FIG. 8.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

1. A solid phase comprising a surface first chemical moiety whichparticipates in covalent carboxyl bond formation with a second chemicalmoiety conjugated to a tag oligonucleotide rendered partially doublestranded by annealing an α-tag oligonucleotide to the tagoligonucleotide to provide a 3′ overhang portion of the tagoligonucleotide wherein the tag oligonucleotide is employed as asubstrate for ligase-mediated covalent bonding to a single-strandedtarget nucleic acid molecule, such that the single-stranded targetnucleic acid molecule is ligated to the tag oligonucleotide, wherein thetag oligonucleotide comprises the second chemical moiety conjugated to aknown oligonucleotide sequence via a molecule comprising mc+n atoms,from about 1 to about 100, wherein m is the number of repeats of size cand n is the number of atoms not included in the repeats.
 2. The solidphase of claim 1 comprising a solid support in the form of amicrosphere, microchip or a glass, plastic or ceramic slide.
 3. Thesolid phase of claim 2 wherein the solid support is a microsphere. 4.The solid phase of claim 1 wherein the covalent carboxyl bond is formedwith an amine group, a thiol group or an acryl group on the secondarychemical moiety.
 5. The solid phase of claim 1 wherein the secondchemical moiety is an amine group.
 6. The solid phase of claim 1 whereinthe α-tag oligonucleotide is labeled with a reporter molecule and isphosphorylated at its 5′ end.
 7. The solid phase of claim 1 furthercomprising a bridging oligonucleotide, said bridging oligonucleotidehaving a nucleotide sequence complementary to the nucleotide sequence ofthe 3′ overhang portion of the tag oligonucleotide and a nucleotidesequence complementary to a terminal end portion of a target nucleicacid molecule.
 8. The solid phase of claim 7 further comprising a targetnucleic acid molecule in ligase-mediated covalent bonding to the tagoligonucleotide molecule anchored to the solid phase.
 9. The solid phaseof claim 6 further comprising a bridging oligonucleotide, said bridgingoligonucleotide having a nucleotide sequence complementary to thenucleotide sequence of the 3′ overhang portion of the tagoligonucleotide and a nucleotide sequence complementary to a terminalend portion of a target nucleic acid molecule.